Calculating MOI for luciferase-transducted cells. - Would the sensitivity of a fluorescent microscope be enough to see the (Apr/27/2009 )
This is likely a very simple question. I just don't know how to Google it, so please give me a brief intro...
I need to measure MOI (multiplicity of infection) for a viral vector carrying a luciferase gene.
I will be infecting adherent cultures in 24 well plates.
I know that for GFP, I would just look at GFP fluorescence in a fluorescence microscope.
But how would I see the Luciferase-expressing cells?
I assume, I need to add a substrate, which will be taken by cells and they would luminescence.
Would the sensitivity of a fluorescent microscope be enough to see the signal?
GFP will glow under normal light.
Anyway, if you are infecting with a pure stock of the virus (i.e. not in conjunction with another virus), then just assess the MOI as you would normally. The GFP doesn't do anything special.
I've only done luciferase measurements on lysed cells, but I assume you can incubate cells with luciferin and detect luciferase activity. However, you can't use a fluorescence microscope for this, as the luciferase produces light on its own. With mice for instance, you can inject the substrate and then detect luciferase activity with a sensitive camera, but I don't know if the signal of your cells will be strong enough to see without such a camers ...